Detection and measurement of blood-feeding activity

ABSTRACT

This invention provides compositions and methods for detection of hematophagous ectoparasitic activity in an enclosure or area. The compositions comprise a reagent or reagents which are reactive against antigens or markers as they appear in the excrement or other ectoparasitic materials. Such markers or antigens may be produced by the ectoparasite itself or may have been introduced into the ectoparasite because of its blood feeding activity. The method of the present invention comprises collecting from the enclosure or area, a sample comprising environmental dust or materials and subjecting the sample to tests for detecting the presence of hematophagous ectoparasitic markers, host markers or both.

This application claims priority to U.S. Provisional application No.60/702,914, filed on Jul. 27, 2005, the disclosure of which isincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to environmental sampling and testing instructures, shelters, or dwellings for markers associated with thepresence of free-living organisms that feed on vertebrate blood.

BACKGROUND OF THE INVENTION

Hematophagy is a behavior of feeding on blood taken from vertebratehosts. It is practiced by about 15,000 species and about 400 genera ofarthropods (Mans, Louw et al. 2002). It has also been reported thatblood-feeding has evolved independently at least six times. In additionto arthropods, members of other phyla including annelids such as wormsand leeches are hematophagous. While independent evolution andwide-spread occurrence of taking blood-meals have resulted inconsiderable biological diversity, there are consistent characteristicspertinent to strategies for hematophagy including: locating andaccessing a suitable blood-containing host, penetrating the integumentand blood circulatory system, counteracting the hemostatic system of thehost, taking a blood-meal and escaping detection so as to survive todigest the meal.

To obtain blood, hematophagous ectoparasites cut or pierce theepithelium and disrupt the integrity of the circulatory system of thehost. During this process of gaining access to blood and feeding onblood, hematophages may inject substances into the host such substancesbeing brought to the symbiotic relationship by the parasite. Theseinjected substances may be either produced by the parasite and assist inblood-feeding or present in the hematophage as a residue of previousfeeding.

Blood-feeding behavior results in numerous new infections in humans peryear and many more in animals. Among the most widely known of theseinfections are cases of malaria resulting from mosquito bites. WhileYellow fever, Chagas disease, Dengue fever, Ebola, and other diseasesare transmitted by tropical insects, human arthropod-borne infectionsalso occur in non-tropical regions. Infectious diseases such asBorrelia, Babesia, Rickettsia, West Nile virus, and various types ofencephalitis infections are found in cold weather climates in theNorthern Hemisphere. Similarly, many non-human species are infected byhematophagous parasites and may serve as alternate hosts or reservoirsfor human pathogens.

Among blood-feeding organisms, arthropods are possibly the best known.Included in this group are arachnids (ticks and mites) and insects,flies, bugs, and lice. Some hematophagous parasites require blood-mealsat every stage of life, such as is the case with ticks, lice, andbedbugs, and others only as adults and in some cases only females areparasitic. Some hematophages spend their entire life on the hosts suchas is the case with head lice. Other species, such as bedbugs, get ontothe source of blood only to feed. The wide biological diversity ofhematophagous organisms presents a challenge to detecting andcontrolling these pests.

Sources of blood for these parasites are vertebrates including, mostimportantly, terrestrial reptiles, birds, and mammals because of theirassociation with humans. Some species of hematophages are opportunisticand will take meals from a variety of host species whereas others maypreferentially or obligately feed on one host species. In many caseshumans are more exposed to hematophagous organisms upon leaving shelter.Well-known blood-sucking insects, such as mosquitoes and biting flies,and ticks, live primarily outdoors. While these parasites may enterhouses, primarily to feed, they usually do not infest human shelters.Typically, arthropod life cycles are more compatible with theenvironment outside of human dwellings and humans are opportunisticallyused as sources of blood.

There are, however, animals that opportunistically share houses withhumans even in developed countries. These organisms are commonly knownas pests. Many arthropod pests enter a dwelling with other pests; forexample, fleas may come in on rodents. It is also possible that pestsenter dwellings on their own, with the humans themselves, or companionanimals such as dogs or cats carry pests into the homes. Infestations byhematophagous ectoparasites can cause annoyance, discomfort, healthproblems, and economic losses. For example, pests in structures otherthan those for human habitation, including but not limited to hen housesfor avian egg production, barns housing domestic animals, mammalianbreeding facilities, or other structures for continued occupation bybirds or mammals, may cause reduced productivity. Once inside, somearthropods are able to establish breeding colonies in the home. Oftenthe homeowner is not aware of such infestations. For example, house dustmite colonies exist in a high percentage of homes in the United Stateswithout the human co-inhabitants being aware of their presence. Theseorganisms feed primarily on human skin flakes that are sloughedconstantly and are a component of house dust. House dust can also serveas the main nutrient source for immature fleas, obligate blood-feedersas egg producing adults.

Pesticide use to control many hematophagous species has undergonesignificant changes with the virtual elimination of “residualinsecticides” (Spielman, Pollack et al. 2001). Subsequently, currentpest management practices have not eliminated hematophagousectoparasites from shelters: school children still are infected by headlice (Pediculus capitas); bedbugs (Cimex lectularius) infest henhousesand are being found with increasing frequency in homes and lodgingfacilities in the United States; fleas (Ctenocephalides sp.) and severalgenera of ticks enter structures inhabited by humans, mice, dogs, cats,and other mammals alone or in combination; and companion animals ofhumans are frequently hosts for are variety arachnid and insectectoparasites. Because modern integrated pest management practices tendtoward more judicious use of pesticides, determining that an infestationexists is a necessary first step in controlling pests and is useful inevaluating the effectiveness of control measures taken. Currently visualinspection either directly or using various collection and magnificationaids is the primary means to determine the presence of pests. Frequentpublication of articles and patents describing new and better ways todetect the presence of pests attests to the fact that current methodsneed improvement.

As mentioned above, blood-sucking parasites may inject substances intothe host during feeding. Compounds of parasite origin have been found tohave various physiological activities including anticoagulation,platelet aggregation inhibition, pain inhibition, and anti-inflammation.Such findings have lead to published studies describing the structureand function of these compounds of possible use as pharmaceuticals.Hirudin, produced by the leech Hirudo medicinalis, has been cloned formass production by recombinant DNA technology and is available fortreating humans as “Refludan” (BERLEX Laboratories, Wayne, N.J.). Thisand other therapeutic applications have lead to an expanding literaturebase in pharmacognosy focused on proteins, especially of salivaryorigin, elaborated by hematophagous invertebrates to assist inblood-feeding (Valenzuela, Walker et al. 1995; Valenzuela, Charlab etal. 1998; Valenzuela, Belkaid et al. 2001). These studies have providedDNA sequences for many salivary proteins which served as useful priorart for the present invention. Salivary biomolecules are injected intothe host circulatory system where the physiological effects occur. Theproteins of hematophage origin are also carried by the blood into theparasite digestive system where they become part of the blood-meal andare subject to degradation by the digestive hydrolytic processes. As aresult, the structure and molecular characteristics of salivary proteinsmay be altered during passage through the digestive system of parasitesand be different from those of the salivary proteins per se or producedfrom recombinant genes.

Vertebrate blood is typically about 80% water. Many hematophagousinvertebrates, especially ectoparasitic species, utilize diuresismechanisms to eliminate much of the water from a blood-meal very quicklyusually as feces (Quinlan, Tublitz et al. 1997). This is necessary formany reasons including improved mobility of the pest to avoid beingdetected and preyed upon or swatted and more efficient digestion. Thevery liquid feces contain compounds that have undergone little digestionduring the short time between feeding and excretion. These feces willcontain biomolecular markers of blood feeding activity derived from boththe host blood and those associated with the hematophagous organisminvolved.

Various types of tests have proven to be reliable and cost-effective fordetecting biomolecules. Those skilled in the art of detecting moleculesof biological origin have developed various methods to offer rapid,easy-to-use, and specific tests with sufficient sensitivity to be usefulfor detecting markers for a wide variety of organisms including but notlimited to bacteria, viruses, parasites, and arthropods in theenvironment. Many of these assays take advantage of the chemicalcomposition and of specific binding properties of biochemicals includingbut not limited to effectors, inhibitors, modulators, and hormonesbinding with receptors; lipids, metals, proteins, hormones beingspecifically bound to carrier proteins; antibodies binding to cellsurface binding domains; and the well-know interaction between antigensand antibodies. Thus, while detection assays are know in the art, suchassays have not been successfully adapted to the detection ofhematophagous ectoparasites that live outside of the body of the host bythe methods and compositions of this invention.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions to detectinfestation of an enclosure or area by hematophagous ectoparasites. Thisinvention is based on the unexpected observation that host specific orparasite specific markers can be identified in environmental dust orother materials collected from the enclosure or area. Therefore anidentification of hematophagous ectoparasitic blood feeding activity canbe made in the absence of visual detection of the ectoparasites.

Accordingly, this invention provides compositions comprising a reagentor reagents to detect the presence of blood-feeding behavior and/or toprovide evidence as to the identity of the hematophagous ectoparasiteand/or the host. The reagents are those that react with antigens ormarkers as they appear in the excrement or other ectoparasitic materials(such as saliva). Such markers or antigens may be produced by theectoparasite itself (ectoparasitic markers or antigens) or may have beenintroduced into the ectoparasite because of its blood feeding activity(host markers or antigens). In one embodiment, the composition comprisesa regent which can react with an ectoparasitic antigen. In anotherembodiment, the composition further comprises a regent which is capableof detecting a host antigen (including, but not limited to, heme,hemoglobin, immunoglobulin or albumin).

The method of the present invention comprises collecting from theenclosure or area, a sample comprising environmental dust or materialsand subjecting the sample to tests for detecting the presence ofhematophagous ectoparasitic markers, host markers or both.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Photograph of bedbug culture on a cardboard substrate showingdark colored spots of feces. Also shown are eggs shells as lightobjects.

FIG. 2 Photograph of bedbug culture debris containing exoskeleton molts(large objects) and small, dark colored fecal matter.

FIG. 3 Photograph of samples cut from bedbug culture substrate cardboardshown after being used in guaiac fetal occult blood test (FOBT).

FIG. 4 FOBT (Biomerica) results with bedbug culture extract (“B”) andoffice dust extract (“D”). Note positive reaction with B and no colordevelopment with D.

FIG. 5 Photograph of FOBT (Immunostics) showing office dust extract onleft and bedbug culture extract on right. A positive reaction is seenwith only with the bedbug extract.

FIG. 6 Photograph of single-use cartridge (IMMOCARE) showing the sampleaddition site (labeled “S”) and the zones labeled “C” where the controlline develops in a valid test. If a line develops in the area labeled“T”, the test is positive and human hemoglobin has been detected. Noline is a negative result. In addition to the cartridge, a vial ofbuffer is supplied to prepare a suspension and extract of the sample.

FIG. 7 Photograph of office dust extract run on a cartridge similar toone shown in FIG. 6. Negative result indicates no human hemoglobin wasdetected.

FIG. 8 Photograph shows result of ImmoCARE IMMOCARE FOBT with bedbugculture debris extract. Strong line at “T” shows a positive resultindicating presence of human hemoglobin.

FIG. 9 Photograph showing bedbug culture debris placed on plastic backedabsorbent paper. Two (2) exoskeleton molts can be seen on left. Fecesand other debris are shown on right side of paper.

FIG. 10 Bedbug culture debris preparation shown in FIG. 9 aftertreatment with luminol (BlueStar).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions to detectinfestation of an enclosure by hematophagous ectoparasites. Thisinvention is based on the observation that biological markers specificfor the host (immediate host or a prior host) or biological markersspecific to the hematophagous parasite can be identified in theexcrement of the parasites. Therefore, this invention makes use of thepresence, in environmental samples, of identifiable biomolecules thatserve as markers of blood-feeding behavior of pests. The methodcomprises two steps: First, a sample representative of the environmentto be tested is collected by any of many methods available including,but not limited to, vacuum collection, swabbing, scraping, shaking, orotherwise causing compounds, dust, and matter in the environment to begathered, collected, or obtained. Such material could be collected suchthat it is suitable for transport. Second, the representativeenvironmental sample is subjected to testing to detect desiredbiomolecule or biomolecules. From the results of the testing usingcommercially available reagents or the reagents of the invention, thepresence or absence of hematophagous ectoparasites can be ascertained.Further, by comparing the levels of the biomolecules to a control level,the degree of infestation can also be determined.

The invention has several advantages over presently available methods ofdetecting ectoparasite infestations. Visual inspection is the mostcommonly practiced method of locating pest infestations. This ispracticed by examining the environment for evidence of the pests, suchas the organisms, molted exoskeletons, and/or fecal matter, by directexamination, combing, trapping, bait and trapping, or vacuuming.Hematophages have developed many different behaviors or traits to enablethem to feed from the vertebrate animal without being killed, removed,or otherwise prevented obtaining blood. These behaviors or traits workagainst the visual methods for establishing the presence of theparasites.

Although hematophagous ectoparasites have different behaviors ofblood-feeding and avoiding detection, it was unexpectedly observed thatsome common attributes could be used for their detection. For example,it was observed that host proteins (or partially degraded portionsthereof), maintained enough integrity to be detectable in the excrementof parasites. Thus, because the pests to be detected by this inventionfeed on blood, based on the observations of this invention, these pestswill be expected to excrete undigested and partially digested componentsof blood in their feces. Because the presence of vertebrate blood in theindoor environment is not normal, the presence of blood, in the absenceof other known causes, can be taken as presumptive evidence of theactivity of blood-feeding. In the present invention standard reagentcompositions or compositions developed as provided herein, can be useddetect any of a myriad of compounds derived from the host blood. Forexample, in one embodiment heme, present in vertebrate blood from allspecies, is detected to look for evidence of blood-feeding in general.In a dwelling inhabited by humans and any of a number of animal speciessuch as dog, cats, birds, ferrets, etc., individually or in combination,detection of heme in the indoor environment indicates the possibility ofblood-feeding pest infestation. Conversely, the absence of heme in housedust or other samples of the environment would provide strong evidenceof the lack of hematophagous ectoparasite infestation. In anotherembodiment, reagent compositions could be used for detection of bloodcomponents, for example blood proteins including but not limited tohemoglobin, albumin, or immunoglobulins, with more specificity thandetecting heme which is present in hemoglobins of all vertebrates.

A second common trait that enables and accompanies blood-feeding isinjection of salivary compounds of parasite origin into the wound in thehost. These compounds, mostly proteins as discussed above, appear in thegut of the blood-sucking pest. As a result of the diversity ofhematophages, these compounds vary highly in structure and compositioneven if not in function. The invention teaches that specificity will beincreased by taking advantage of this biochemical diversity.

The method and selected reagents of this invention could be used todetect hematophagous ectoparasites including bedbugs and other bugs,lice, mites, fleas, mosquitos, flies, and ticks Those species ofparticular interest are those that can infest dwellings used by theirhosts. In one embodiment, bedbugs (Cimex lectularius) and Pediculushumanus (human head and body louse) are detected by the method of thisinvention.

Broadly, it is an object of the instant invention to providecompositions of prescribed reagents to detect the presence ofblood-feeding behavior and to also provide evidence as to the identityof the ectoparasitic hematophages involved. The reagents will be thosethat react with antigens or markers as they appear in the excrement orother ectoparasitic materials (such as saliva). Such markers or antigensmay be originating from the ectoparasite itself (ectoparasitic markersor antigens) or may have been introduced into the ectoparasite becauseof its blood feeding activity (host markers or antigens). Non-limitingexamples of such markers or antigens are those that are present in theexcrement and/or saliva of the ectoparasite—whether due to beingproduced by the ectoparasite or derived from a host on whom theectoparasite has previously fed. In one embodiment, the compositioncomprises a regent which can react with an ectoparasitic antigens. Inanother embodiment, the composition further comprises a regent which iscapable of detecting a host antigen.

In the present invention, testing may be performed on an environmentalsample depending on the information that is to be obtained. For example,testing may be directed to detecting all blood feeding activity (such asdetection of heme in the environmental sample), detecting blood feedingon humans (such as by using probes specific for human blood proteins,detecting for the presence of all blood feeding pests (such as bydetecting salivary proteins of pest-origin), or the presence of specificblood feeding pests (such as by detecting species specific salivaryproteins). Those skilled in the art will recognize that any combinationor routine modification of the above can be used depending upon theinformation desired.

The method of the present invention can be useful in detectingblood-feeding activity in such places as: multi-room homes; hotels,motels, hostels, dormitories, boarding houses, shelters, and any otherfacilities in which people live or otherwise occupy; transportationrelated enclosures such as cruise ships, railway sleeping cars,airplanes; and animal housing enclosures such as henhouses, indoorrodent colonies, vivaria, and non-carnivorous animal breedingestablishments. In multi-room establishments, samples, such as dustcaptured by vacuuming, can be collected from each room and tested forthe presence of, for example, hemoglobin. Those rooms that test positivecan be subjected to treatment or additional testing.

In one embodiment, the invention can also be used to discriminatebetween blood feeding organisms of different species or blood feedingorganisms feeding on more than one species. Therefore, the method of thepresent invention could be used in structures where more that onevertebrate species may be present either intentionally or byhappenstance. In these cases the composition of the testing reagent canbe selected to be specific for detecting hemoglobin, or other bloodsubstance, of the species of interest. For example, a number or reagentsare available (or can be produced by standard techniques) which reactspecifically with human and which do not react with hemoglobins fromcats, dogs, or rodents. Such reagents can be used to specifically testfor blood feeding parasite activity on humans. Alternatively, a reagentspecific for canine hemoglobin could be used to differentiate betweenparasites feeding on dog blood from those feeding or humans, cats, androdents. Such compositions can be useful in homes occupied by people andtheir vertebrate pets and in barns occupied by a colony of domesticanimals and also possibly by vertebrate pests. In such a test, thepresence of blood-feeding activity and the host could be determined byusing reagents similar to those currently used to obtain speciesidentity of blood stains in forensic serology. For example, recently, ahuman hemoglobin specific rapid field-use forensic test has beenintroduced by BlueStar, ROC Import, Monte Carlo, Monaco. This test givespositive results for human hemoglobin while being negative for bloodfrom non-primate animals. The same art has been utilized by several ofthe immunological FOBT described herein to allow for detecting humanhemoglobin without giving false positive reactions with non-humanhemoglobin.

A second embodiment of the present invention is to test for the presenceof a marker of parasite origin in samples. This method is preferred insituations in which detecting compounds derived from host blood mightnot be sufficient to confirm the infestation. This method would also beuseful in determining the identity of the pest. It is important inselecting the composition of the reagents to practice this embodiment toidentify the structure and binding properties of the biomolecule orbiomolecules to be detected as they exist in the environment dustsamples. These properties may or may not differ from those of the nativemolecule as produced in the digestive system, including salivary glands.Therefore, in spite of knowing the gene sequence and protein structure,the properties of the biochemicals of interest may be altered by passagethrough the digestive system and release into the environment. This arthas been practiced to increase specificity of immunoassays including theFOBT produced by Care Products, Waterbury, Conn. In this test,antibodies selected for use are specific to hemoglobin released into thedistal gut and not to the more extensively degraded hemoglobin emanatingfrom mouth, stomach, and proximal gut locations.

A third embodiment is detecting in the environment dust samplescomprising excrement of hematophagous ectoparasites, one or more markersindicative of a previous feeding of the pest. For example, a tickbecomes infected with Borrelia, the Lyme Disease organism, by feeding ona small rodent infected with the organism. During a subsequent feedingon humans, the infected tick may inject Borrelia microorganisms into thebloodstream of that host with saliva. Some of the Borrelia organismsthus injected can then be ingested by the ectoparasite that injectedthem. Therefore, the excrement of the ecotoparasite may contain not onlythe markers for the immediate past host (in this example a human), butalso a marker obtained by the ectoparasite from a prior host (in thisexample Borrelia). In manner similar to that described for bloodcomponents and salivary compounds, compounds of microbial origin may befound in excreted materials. Microbes, molecules, or markers derivedtherefrom are then available for detection in environmental samples.Such tests could be very valuable for epidemiological studies wherecatching organisms is not practical.

In the method of the present invention, collection of an environmentalsample is the first step in detecting presence of hematophages. Theterms “environmental dust sample” or “environmental dust” as used hereinrefers to materials collected from an enclosure that can be expected tocontain the excrement, which in turn contains markers of blood feedingby pests. Such environmental dust sample or environmental dust maycomprise materials including flakes and smears from diuresis eliminationof water by pests described previously; shells, casts, casings, and anyother remnants of the blood-feeding pest bodies or eggs; and fecalmatter containing markers as described herein. Any method by which anenvironmental sample or environmental dust can be collected, can beused. One well-established method is the vacuum trace evidencecollection practiced in crime scene investigations. These systems arecommercially available (a vacuum evidence collection kit is offered by,for example, 3M, Minneapolis, Minn.) The utility of these systems hasbeen well accepted in criminal investigative work. Disposable filtersand sealable plastic containers, designed to maintain sample integrityand chain of custody, might be well suited to collect samples in theinstant invention. In addition, other sample collection methods to beused include collecting dust accumulated on home HVAC filters; filtersin room air conditioners in hotels and motels; dust accumulated on ornear ventilation fans in barns, stables, henhouses, etc.; dust collectedby electrostatic particle precipitators; and samples collected by dampmopping non-porous floors such as tile floors in schoolrooms. Specificsample collection methods are not necessary to practice the inventionbut the validity and therefore the utility of the results are dependentupon collecting and maintaining the integrity of a representativeSample. For example, in a room with a suspected blood-feeding pestinfestation, it might be deemed useful to vacuum the whole room toscreen for markers; to manually collect dark-colored flakes around thehead of the bed; to vacuum only around the head of the bed to look morespecifically for bedbugs due to their feeding habits; to lift materialswith water-dampened swabs; or possibly all or any combination ofcollection methods. All samples thus obtained could be tested for any orall markers depending upon judgment of the persons involved, costs,desired thoroughness, etc. All or a portion of the collected materialsin the environmental dust sample is then used for detection of bloodfeeding activity.

The second step in the invention is to test for marker or markersindicative of blood feeding activity. The invention provides forselecting reagents depending upon the specific inquiry. For example,reagents can be used for detection of heme, which is present in allvertebrate blood and is expected to be present in the excrement of allblood-feeding parasites following blood feeding activity. The inventionalso provides for species specific screening for a marker or markerssuch as using monoclonal or polyclonal antibody based immunoassays todifferentiate human hosts from animal hosts, for example. Examples ofsuch reagents include antibodies (including antigen binding fragmentsthereof) to species specific blood proteins as described herein forhuman hemoglobin and IgG. The invention also provides for parasitespecific screening, that is, testing for markers that differentiate aparasite species from others without providing information on the hostspecies. The invention further provides for combinations of reagents ortest components designed to provide relevant information for theconditions encountered. Further, the method provides for detectingmarker or markers released into the environment by blood-feeding saidmarker or markers being present in the pest as a result of previousfeeding or feedings.

Detecting components of the blood of the host or those derived from theparasitic organism may be accomplished by direct chemical methods suchas guaiac oxidation, cyanmethemoglobin tests or more specificligand-binding assay, such as immunoassay, nucleic acid based detectionmethods or other suitable methods.

Examples 1, 2 and 3, provide details on the methods to detect vertebratehemoglobin in hematophagous insect feces using chemical reactionsinvolving the metalloporphyrin moiety present in hemoglobin from allvertebrate species. One method is based on reaction between heme ofhemoglobin with guaiac after which a developer solution containinghydrogen peroxide is added. If hemoglobin is present, the guaiac isoxidized, turning the detection reagent blue. In addition, several othermethods that might be used to detect hemoglobin in samples collectedcould be used. One of particular interest, because of itswell-established sensitivity would be luminol spray used successfully byforensic scientists to visualize bloodstains not visible to the humaneye. Samples can be collected by vacuum onto a barrier filter membraneas described herein and as practiced in forensics. The membrane can beremoved and placed into a dark chamber in view of a sensitive camera.Either film or digital photography could be employed. A luminol solution(such as from BlueStar), can be sprayed onto the membrane and samplematerial associated thereto and chemiluminescence, if any, would berecorded by the camera suitably setup and activated.

Liquid Samples, such as those obtained by mopping an enclosure, or awater extract of a dust sample can also be used. The use of thecyanomethemoglobin method (with Drabkin's reagent described in Drabkin,D. L., Austin, J. H. Journal Biological Chemistry 112, 51, (1935)) todetect and measure hemoglobin is well established in diagnostic andveterinary medicine and can be used in this embodiment by adding thesample to the reagent and test for the development of the specifiedcolor.

In Examples 4 and 5, immunological determination of human hemoglobin inhematophagous arthropod feces is disclosed. These examples demonstratethat proteins, as exemplified by the globin portion of hemoglobin,present in the blood-meal retain antigenic characteristics that enabletesting for the protein using immunochemical reactions. In addition tothe immunological fecal occult blood test (iFOBT) other antigensincluding animal blood proteins and salivary proteins of parasiteorigin, and antigens associated with infectious disease causative agentssuch as malaria, West Nile virus, trypanosomiasis, etc, can also beused. To accomplish this, commercially available antibodies (such asdirected against canine hemoglobin or chicken hemoglobin) can be used orantibodies specific for markers of interest may produced usinghematophages or parts or products of hematophages such as feces.Commercially available antibodies, raised against intact proteinsisolated from host blood, can also be used as reagents of the invention.Alternatively, or additionally, antibodies can also be generated againstproteins present in the feces. This will address the issue ofdegradation of antigenic epitopes during the digestive process. Asimilar approach can be used for salivary proteins produced by thehematophagous arthropod. Once antibodies are obtained eithercommercially or raised against the proteins or peptides, the antibodycan then be validated for use by establishing reactivity with antigensin feces. Such antibodies can be employed in any of various immunoassaytechniques available to those skilled in the art including but notlimited to immunochromatographic or lateral flow techniques, ELISA,double diffusion, particle agglutination, immunoprecipitation, laser andnon-coherent light nephalometry, and immunoinhibitory assays. Theselection of the technique will be made based on the requirements of theSample, the information being sought, and the venue in which the testwill be performed.

Examples 6 and 7 describe results obtained with environmental samplescollected from a multiplicity of rooms in hotel building operatingcommercially. In addition to testing for hemoglobin using methods ofpreceding Examples, the versatility, adaptability, and broadapplicability of the present invention is disclosed by successfullytesting for IgG, a blood protein, in the environmental samples (Examples8). Examples 9 describes a combination of immunological and chemicaldetection methods. Example 10 describes results obtained from a samplecollected from non-human housing structure.

Examples 11-13 demonstrates the feasibility of generation of antibodiesto salivary antigens of hematophagous ectoparasites such that theantibodies can be used for detection of the parasites in parasiticexcrements or environmental dust samples from enclosures.

The following Examples describe the preliminary results obtained inexperiments conducted prior to filing this application.

EXAMPLE 1 Detecting Heme-Iron in Bedbug Culture Substrate

Cultures of bedbugs (Cimex lectularius) were maintained in glass jarscontaining a pressed paper cardboard substrate (FIG. 1) at roomtemperature and were allowed to feed at 7 to 14-day intervals on humanblood. The bedbugs were allowed access to human skin through a 200-meshnylon screen attached over the top of their jars. The skin was allowedto be in contact with the mesh for at least 10 minutes with each jarduring which time the bedbugs fed to repletion. Cardboard substrate wasremoved from each jar at least 10 days after feeding, put into plasticbags, sealed, and stored in the freezer at temperatures below −10° C.until processed.

Fecal occult blood tests (FOBT) of two different peroxidase activityvisualization chemistries were used from three different commercialsuppliers (guaiac from Immunostics, Inc., Ocean, N.J. and Care Products,Waterbury, Conn. and 3,3′,5,5′-tetramethyl benzidine (TMB) fromBiomerica, Inc., Newport Beach, Calif.) were used to qualitativelydetect heme. Samples tested were bedbug culture debris (an example ofwhich is shown in FIG. 2) and pieces of the cardboard cut from thesubstrate (such as shown in FIG. 3).

Extracts were made by putting bedbug culture debris into vials of buffersupplied by Care Products for the immunologic FOBT and shaken for about5 minutes. Two drops of these extracts were placed on EZ Detect pads(Biomerica, Inc.) employing the TMB chemistry and processed according tomanufacturers instructions. FIG. 4 shows the results with cleardevelopment of color (positive result) with the bedbug culture extract.FIG. 5 shows the results of the same extracts run with Immunostics'guaiac FOBT. In addition, pieces of the culture substrate, such as shownin FIG. 3, were placed on the paper of both the Immunostics and CareProducts guaiac FOBT. Upon addition of the developing reagent, positiveresults were observed. A molted exoskeleton gave a negative result inguaiac FOBT.

It can be concluded from these results that the peroxidase activity suchas that of hemecan be detected in the feces of bedbugs

EXAMPLE 2 Detection of Blood in Bedbug Cultures Using Luminol

Debris collected from bedbug cultures as in Example I was placed onabsorbent plastic-backed paper (FIG. 9) and sprayed with a commerciallyavailable luminol blood visualization solution (BlueStar, ROC Import,Monte Carlo, Monaco). The resultant chemiluminescence was seen to beassociated with the debris and not with the molted exoskeletons (FIG.10). This visualization technique can be used as in initial step toidentify individual or clusters of particles emitting light to beisolated and subjected to further testing to confirm hematophageactivity.

EXAMPLE 3 Detection of Blood in Bedbug Cultures Using Urine Test Strips

Debris collected from bedbug cultures as in Example I was dissolved inwater and tested for the presence of blood using commercially availableurine dipsticks (such as from Roche or Bayer). With each test thepresence of detectable blood was indicated by positive reaction on theappropriate pads.

EXAMPLE 4 Detection of Human Hemoglobin in Bedbug Cultures withMonoclonal Antibodies

The culture debris extracts from Example I were also tested withIMMOCARE (immunological FOB from Care Products, Inc. Waterbury, Conn.).The suspensions were added to cartridges (FIG. 6) provided with the kitand allowed to develop according to the instructions. The results wereread per the instructions and are shown in FIGS. 7 and 8. The bedbugculture debris extract clearly gave a positive result indicating thatsufficient epitopes were present of the hemoglobin in the feces to reactwith the monoclonal antibodies in the iFOBT.

EXAMPLE 5 Detection of Human Hemoglobin in Bedbug Cultures withPolyclonal Antibodies

The culture debris extracts from Example I were tested for reactivitywith commercially available polyclonal raised against human hemoglobinby direct and sandwich immunoassays. For direct detection, 20 μL ofextract was applied to nitrocellulose membranes and washed to removeunbound materials. The membranes were blocked with 20% horse serum inbuffered saline and washed 3 times. Spotted membranes were incubatedwith 2% horse serum-saline alone or containing polyclonalanti-hemoglobin labeled with horseradish peroxidase (HRP) or alkalinephosphatase (AP) and washed to remove unbound enzyme-labeled antibody.Any enzyme activity bound via antibody to antigen present in the debrisextract bound to the membrane was visualized with the appropriatesubstrate. Clearly visible colored spots were observed when spottedmembranes were exposed to enzyme labeled anti-hemoglobin and theappropriate substrate while no visible reaction was observed when eithersubstrate was exposed to spotted membranes not allowed to react withantibody.

Sandwich immunoassays for hemoglobin in culture debris by immobilizingpolyclonal anti-hemoglobin to nitrocellulose membranes, washing, andblocking as described above. The capture antibody membranes were thenallowed to incubate with culture debris extract, washed, and exposed toenzyme labeled anti-hemoglobin. After visualization with enzymesubstrate, the presence of hemoglobin in the culture debris wasindicated by developed color.

Bedbug culture debris was incubated with affinity purified polyclonalanti-hemoglobin to occupy epitopes necessary for positives reaction iniFOBT assays of Example 4 or immunoassays of Example 5. In all cases,prior treatment of the antigen with anti-hemoglobin prevented detectionof hemoglobin by the immunoassays described in the cited Examples.

EXAMPLE 6 Detection of Hemoglobin in Environmental Samples—SampleCollection

Examples 1 through 5 demonstrated that evidence of blood feeding byhematophagous organism can be obtained from the organisms' fecal debris.To establish the utility of these methods for detecting naturallyoccurring blood feeding by testing environmental samples, samples werecollected from offices, hotel rooms, and homes. Samples were collectedby methods described herein including using standard vacuum cleaners tocollect vacuum bag dust, using handheld portable vacuum devices,manually removing dust collected on filters of HVAC systems, airconditioners, and fans, and collecting dust manually or by wiping. Dustsamples thus collected were used directly without further treatment orextracted with any of a variety of aqueous solutions to prepare liquidsamples in the examples that follow.

EXAMPLE 7 Samples from Office and Hotel Rooms—Testing for EnvironmentalHemoglobin as Evidence of Blood Feeding

Vacuum dust collected by vacuuming and manually removed from airconditioner filters from individual rooms were tested with luminol byspraying the test solution onto dust samples as collected. Some vacuumdust samples gave positive chemiluminescence with various densities oflight emitting particles per volume of dust while other samples gave novisible chemiluminescence. Samples of dust from filters of airconditioners, such dust being a capture or aerosolized particles overtime, were uniformly negative for chemiluminescence when tested withluminol. These results indicate that the luminol test has sufficientsensitivity to be useful in the present invention.

The samples were also tested for hemoglobin using FOBT and test stripsgiving results consistent with the luminol results. These results alsoindicate that testing for the heme moiety is useful for screening, thatis to eliminate sites without detectable blood-feeding activity andtherefore possible infestation from further investigation at a giventime.

EXAMPLE 8 Samples from Hotel Rooms—Testing for Human IgG as Evidence ofBlood Feeding

Samples from bedbug cultures and environmental samples were extractedwith dilute (0.001 N) sodium hydroxide, neutralized by addition of 0.2 Mpotassium phosphate in 0.9% NaCl, and tested at various dilutions forthe presence of human IgG, a blood serum protein, using a commerciallyavailable enzyme linked immunosorbent assay (ELISA) test kit(ZeptoMetrix Inc., Buffalo. NY). Positive results for IgG inenvironmental samples as well as Cimex culture debris indicated thepotential utility of testing for blood components other that hemoglobinfor screening or detecting blood feeding activity. These resultsdemonstrate that that proteins other than the globin moiety ofhemoglobin remain sufficiently structurally intact after the digestiveprocess by Cimex to be detectable by standard immunological reagents andmeans. The result also provide support for the idea that the methods andcompositions of the present invention can be designed to be applicableto a variety of testing requirements.

EXAMPLE 9 Detecting Hemoglobin by Combined Immunological and ChemicalMethods

Culture debris and environmental samples from previous examples werefurther tested with an immunoassay and luminol combination method. Testmembranes to which monoclonal antibodies specific for human antibodieswere removed from the iFOBT devices previously described. In addition,approximately 1 microgram of polyclonal anti-human-antibody obtainedcommercially from Bethyl Labs, Montgomery, Tex., was immobilized on anitrocellulose membrane strip. Both sets of antibody test strips werewashed in PBS and coated with 10% normal horse serum in PBS for 30 min.Hotel room and culture debris extracts, also in PBS, were added to thestrips for 2 hours at room temperature. Test strips not exposed to theextracts were included in the experiment as negative controls. Afterincubation with the samples or control, all strips were washed 3 timewith PBS. Each strip was sprayed with luminol solution as previouslydescribed and examined for chemiluminescence which was clearly observedwith the antibody strips exposed to samples. In the control strips nochemiluminescence was observed at the location of the immobilizedantibody. This experiment demonstrates that analytical methods can beused alone or combined to depending upon the testing needs.

EXAMPLE 10 Samples Chicken Houses—Environmental Samples

Samples of dust from the floor, trays, fans, and fan filters werecollected from structures housing chickens. Some of the birds were inindividual cages and some were in groups of several dozens free to moveabout contained in open rooms. Positive chemiluminescence and dip sticktests were obtained with samples but all tests using immunoassaysspecific for human hemoglobin were negative. These results demonstratethe capability to design test reagents and systems for detectingectoparasitic blood-feeding on animal hosts and the ability todifferentiate such activity from that occurring with human hosts.

EXAMPLE 11 Antibodies Against Hematophage Antigens

Blood was drawn by a healthcare professional from a human volunteer whohad been bitten by Cimex repeatedly for more than 3 years and who hadconsented to provide blood and have it tested for reactivity withsamples obtained as described herein. Plasma was prepared, separated andfrozen. To determine the presence of antibodies against antigens presentin bedbug culture debris, a debris extract was applied to nitrocellulosemembranes as in Example 5, washed, blocked with 2% normal horse serum inPBS, and exposed to human plasma from above at various dilutions. Themembranes were then washed and allowed to incubate with alkalinephosphatase labeled anti-IgG (human). The presence of antibodies toantigens present in the bedbug culture debris was visualized by exposingthe membrane so treated with substrate for alkaline phosphatase. Thepresence of color was observed at antiserum concentrations higher thanat a 1:500 dilution and was not observed on membranes not exposed to thehuman antibody solutions. These results indicate that antibodiesproduced in response to exposure to hematophage saliva can be used fordetecting the presence of pest excrement. Such presence could indicatepast or present blood feeding activity.

EXAMPLE 12 Collection of Cimex Saliva

Saliva was collected from bedbugs immobilized with adhesive tape bypositioning the mouthparts of the insect into a capillary tube. Asolution of pilocarpine in alcohol sufficient to induce saliva excretionin the individual being used was applied to the head of the insect. Thesaliva thereby secreted was collected in the capillary, transferred tovials, sealed, and frozen until used.

EXAMPLE 13 Testing of Reactivity of Antibodies Against HematophageSalivary Antigens

Human antibodies preparation collected as described in Example 11 wastested with Cimex saliva described in Example 12 by immobilizingproteins on to nitrocellulose membranes, blocking and washing aspreviously described. The membranes were incubated with human antibodiesat dilutions varying from 1:50 to 1:1000 in PBS containing 2% normalhorse serum and the reactivity was detected withenzyme-labeled-anti-human IgG as previously described. Color developmentindicated reaction between the saliva sample and human antibodies. Thereactivity was similar to that observed in Example XI and was consistentwith the presence of immunologically detectable hematophage specificmarkers in saliva. This example demonstrates that antibodies generatedfrom individuals who have been bitten by the hematophagous parasites(and therefore exposed to the salivary proteins of the parasites) can beused for the detection of parasites in an environmental sample. Thuspolyclonal or monoclonal antibodies from individuals (humans or animals)could be obtained following exposure to the parasitic antigens such assalivary proteins—whether by being bitten by the parasite or otherconventional immunization procedures. Because the volunteer donorreported redness and itching as a result of bedbug bites—a physiologicalreaction associated with an immunoreaction to substances injected insaliva—these results support the conclusion of the inventor that theinvention will have utility in testing for any blood-feedingectoparasite to which the host mounts an immune response.

REFERENCES

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1. A method of determining a likelihood of blood feeding activity by ahematophagous ectoparasite in an enclosure inhabited by individuals of ahost species, wherein the hematophagous ectoparasite is a bedbug, themethod comprising the steps of: a) collecting a sample of environmentaldust from the enclosure; b) detecting for the presence or absence in theenvironmental dust sample of a marker selected from the group of markersconsisting of host hemoglobin, hematophagous ectoparasite salivaryprotein antigens, and combinations of host hemoglobin and hematophagousectoparasite salivary protein antigens, which host hemoglobin marker orhematophagous salivary protein antigen marker is detectable in excrementof the hematophagous ectoparasite, wherein detecting the presence of themarker in the environmental dust sample is indicative of a likelihood ofblood feeding activity by the hematophagous ectoparasites feeding on theinhabitants of the host species in the enclosure and wherein the absenceof the marker in the environmental dust sample is indicative of alikelihood of a lack of blood feeding activity by the hematophagousectoparasites feeding on the inhabitants of the host species in theenclosure.
 2. The method of claim 1, wherein the environmental sample iscollected by vacuuming, swiping or mopping.
 3. The method of claim 1,wherein the host species is selected from the group consisting of human,dogs, cats, rodents and chickens.
 4. The method of claim 1, wherein theenclosure is inhabited by humans.
 5. The method of claim 1, wherein theenclosure is inhabited by non-human animals or birds.